There is rapidly increasing interest in developing molecular imaging approaches that enable traditional radiological imaging techniques to obtain a wide range of information about molecular and cellular processes that occur in normal and diseased tissue. A range of information is considered important such as the ability to monitor cell migration, the development of reporters that enable imaging of gene expression, the development of robust strategies to image receptors, and the development of environmentally sensitive agents that can be used to detect the presence of specific enzymes or monitor changes in ion status. The long term goals of this work are to develop strategies that enable MRI contrast that is sensitive to a wide range of molecular and cellular processes. This work builds on over 15 years of work where we have demonstrated the first MRI strategy for detecting gene expression, the first MRI approach for monitoring a surrogate of calcium influx, the first MRI approach for performing neuronal track tracing, and the first MRI approach for monitoring the migration of single cells in vivo. These all represented initial reports by any radiological imaging technique which enabled these processes to be measured. These techniques are finding widespread application to imaging pre-clinical models of a broad range of diseases. Over the past year we have made progress in all of the specific aims. Aim 1: Develop iron oxide based contrast for labeling and imaging the migration of endogenous neural stem cells. Over the past few years we have demonstrated the unique advantages of micron sized iron oxide particles for MRI of specific cells. Single cells can be detected and indeed, single particles within single cells can be detected. The main paradigm for MRI of cell migration is to label cells ex vivo and monitor migration after transplantation into an animal. These studies have traditionally required very efficient labeling using nano sized particles. The ability to detect a single particle enables inefficient labeling strategies. In particular we have demonstrated that injection of particles into the ventricles of the rat brain enables particles to be taken up by neural precursors in the subventricular zone and MRI can monitor the migration of cells to the oflactory bulb. Over the past year we have used the ability of MRI to count new cells in the bulb to address the issue of whether odor exposure alters migratory patterns of these cells. PReviosu work by a number of groups has led to conflicting results, potentially due to bias analysis of histological data. Rats were expsoed to specific odors for two weeks and cells counted throughout the bulb. No changes in cell counts were detected in any region of the bulb except in the mitral cell layer where the number of new cells doubled. This increase occurred primarily in regions known to be activated by the odor. These cells have been shown to be interneurons. The role of these cells in olfactory processing and whether other odor specific behaviors effect cell number in this region of the brain will be assessed. Aim 2: Apply microfabrication techniques to manufacture unique metal structures that may be valuable for MRI contrast. Iron oxide particles commonly used for MRI are very potent contrast agents enabling detection of single mciron sized particles. However, due to bulk phase manufacture of particles they are not very uniform and they do not contain very high content of metal. A solution to this problem is to use modern microfabrication techniques to manufacture metal based, micron sized contrast agents. To begin this work we have explored a variety of approachs to microfabrication of MRI contrast agents. We have demonstrated that precise definition of shapes and spacing of microfabricatedstructures leads to novel MRI agents. As expected micron sized microfabricated nickel structures are very potent MRI contrast agents. Microfabrication gives us a great deal of flexibility to make structures that may have novel uses. For example, particles spaced at distances much smaller than an MRI voxel can be distinguished and water associated with properly designed structures can be distinguished. Over the past year we have demonstrated that micron sized cylinders are very useful for distinguishing structures. Furthermore, we have made simple gold coated iron discs that are about 10 times better than traditional micron sized iron oxide particles. Over the next year we plan to begin to label cells and transplant into animal sto show the in vivo efficacy of these microfabricated structures. Aim 3: Develop novel delivery mechanisms to extend the applicability of manganese enhanced MRI. Over the past ten years we have demonstrated the remarkable utility of the manganese ion for MRI contrast. Manganese ion enters cells on ligand or voltage gated calcium channels and so can be used as an MRI agent to monitor calcium influx. Once inside of neurons, manganese will move in an anterograde direction and cross functional synapses enabling neuronal networks to be imaged with MRI. Finally, manganese given systemically gives cytoarchitectural information about the rodent brain. These successed have us interested in broadening the ways in which manganese ion can be delivered to cells. Over the past couple of years we have made transferrin-manganese complexes. When bound to transferrin manganese is a poor MRI contrast agent. However, when transferrin is taken up by cells it can release manganese which is then trapped intracellularly. Thus, transferrin manganese is an agent that monitors the successful endocytosis of the transferrin by its receptor. Experiments in hepatocytes and in brain demonstrate that this strategy is succesful and gives efficient contrast. We have managed to get similar effects with MnOxide based nanoparticles. At pH 7 MnO is insoluble and a very weak contrast agent. At low pH, as found in endosomes/lysosomes these particles dissolve greatly increasing MRI relaxation effects. Over hte apst year we have demonstrated that a silica coat on these particles delays dissolution for up to four hours both in vitro and in vivo. Particles injected into the brain had slower rates of contrast development and neuronal tracing then did injection of MnCl2. This opens the possibility of making coatings that can be enzymatically degraded enabling specific in vivo assay of these enzymes. This strategy is limited to endosomal/lysomal enzymes but hold promise for increasing the specificity imaging agents. Aim 4: Develop strategies that enable cellular processes to alter the relaxivity of MRI contrast agents. In specific aim 3 we demonstrated a way in which a normal biological process (endocytosis of transferrin-Mn) can alter the effectiveness of an MRI contrast agent. It would be very exciting to find ways in which this can occur which are sensitive to other biological processes. To this end we have begun to explore ways in which the microfabricated particles produced under Aim 2 can be modulated. It is clear that modulating water exchange alters contrast properties and practical approaches to achieving this aim will be explored over the coming year.